iRT peptides/protein as global standard

iRT peptides/protein as global standard Juan C. Rojas E.  2022-01-06

Hi all,

I spiked a protein to my sample before performing the digestion. Then I used the peptides of this protein to define the custom iRT peptides.

The main purpose of the protein spike was to control variance of the digestion procedure so I would like to set it as a global standard. However, when I tried to set the peptides of this protein as a global standard the option is greyed out (image attached). What is the reason behind this? Besides removing the iRT predictor (and with it the annotated iRT peptides) what else could I do to consider these peptides as global standards?

Would you suggest I use all the peptides of the protein or just a few intense ones? If the second, how many?

As always, thank you for your help and time.
Juan C.

PS: I am using Skyline

Nick Shulman responded:  2022-01-06
If you want to use the same peptides as both iRT standards and as global normalization standards, the recommended thing to do is to have two copies of the peptides in your document.

That is, you can select all of the iRT standards in the Targets tree, and then use Edit > Copy and Edit > Paste to get a second copy of those peptides.
The first occurrence of the peptides will be the ones with the clock icon next to them which you will not be able to change the Standard Type of. You can right-click on the second copies and mark them as Global Standards.

In terms of which peptides to use as Global Standards, if you have technical replicates, I would recommend choosing different sets of peptides as Global Standards and figure out which set of those results in the lowest CV within those technical replicates. You can either use the CV histogram in the Skyline ("View > Peak Areas > CV Histogram") to see those CVs, or you can use the Pivot Editor in the Document Grid which is detailed here:

-- Nick
Juan C. Rojas E. responded:  2022-01-07
Hi Nick,

Thank you very much for the quick response. I will proceed with your suggestions.

- JC